Human cytomegalovirus neutralising antibodies and use thereof

ABSTRACT

The invention relates to neutralizing antibodies which are specific for human cytomegalovirus and bind with high affinity as well as immortalized B cells that produce such antibodies. The invention also relates to the epitopes that the antibodies bind to as well as the use of the antibodies and the epitopes in screening methods as well as the diagnosis and therapy of disease.

This application is a Continuation of U.S. patent application Ser. No.13/619,305, filed Sep. 14, 2012, now U.S. Pat. No. 8,545,848, which is aContinuation of U.S. patent application Ser. No. 13/092,364, filed Apr.22, 2011, now U.S. Pat. No. 8,309,089, which is a Continuation of U.S.patent application Ser. No. 11/969,104, filed Jan. 3, 2008, now U.S.Pat. No. 7,955,599, which claims priority to British Patent ApplicationNo. GB 0700133.2, filed Jan. 4, 2007. The entire contents of each ofthese patents and patent applications, along with all documents citedtherein, are hereby incorporated by reference.

This invention relates to antibodies having specificity for humancytomegalovirus, suitably monoclonal antibodies having that specificityand immortalised B cells that produce such monoclonal antibodies. Theinvention also relates to the epitopes that the antibodies bind to aswell as the use of the antibodies and the epitopes in screening methodsas well as the diagnosis, prophylaxis and therapy of disease.

Human cytomegalovirus (hCMV) is a widely distributed pathogen that maycause severe pathology in immunosuppressed adults and upon infection ofthe fetus and has been implicated in chronic diseases such asatherosclerosis. hCMV infects multiple cell types including fibroblasts,endothelial, epithelial and hematopoietic cells [1]. In vitro propagatedattenuated strains of hCMV, which are being developed as candidatevaccines, have lost the tropism for endothelial cells, while retainingthe capacity to infect fibroblasts [2]. Recently two viral glycoproteincomplexes have been shown to control the cellular tropism of hCMV. Acomplex of gH, gL and gO is required for infection of fibroblasts, whilea complex of gH, gL and proteins encoded by the UL131-UL128 genes areresponsible for infection of endothelial cells, epithelial cells anddendritic cells [2-8].

Hyperimmune globulins are already commercialised for the prophylaxis ofhCMV disease associated with transplantation and recent evidenceindicates that they have therapeutic effect in pregnant women [9]. Thistherapeutic approach is limited by the low amount of neutralisingantibody that can be transferred and for this reason the availability ofhuman antibodies (such as human monoclonal antibodies) with highneutralising capacity would be highly desirable. However the target ofhCMV neutralising antibodies remains to be established. Previous workidentified gB and gH as potential targets. However, a humanised antibodyto gH (MSL 109) did not show any significant effect in a clinical trial[10, 11]. All neutralising antibodies described so far had lowneutralising capacity as they neutralise hCMV infection only at highconcentrations. For instance, the antibody MSL-109 only displayed a 50%neutralising activity at a concentration of 10 μg/ml, a fact that mayexplain the lack of an in vivo effect [11]. The neutralising potency ofanti-hCMV antibodies is typically measured using fibroblasts as targetcells. However, hCMV is known to cause pathology by infecting other celltypes such as endothelial, epithelial cells and leukocytes. There do notappear to be any monoclonal antibodies available that would be capableof neutralising with high potency infection of non-fibroblast targetcells. The recently described neutralising antibodies to UL128 and UL130also showed very low potency in neutralising infection of endothelialcells [7].

There is therefore a need for the production of neutralising antibodiesagainst hCMV as well as the elucidation of the target to which suchantibodies bind.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a SDS-PAGE which demonstrates that human monoclonalantibodies (1) 1F11 and (2) 2F4 precipitate complexes of hCMV proteins,whereas irrelevant IgG does not.

FIG. 2 shows a FACS analysis which demonstrates that human monoclonalantibodies (A) 1F11 and 2F4 and (B) 5A2 and 9A11 recognise aconformational epitope made by UL130 and UL131A gene products.

FIG. 3 shows the nucleotide and amino acid sequences of the heavy andlight chains of 1F11, SEQ ID NOs: 9 and 10 and SEQ ID NOs: 7 and 8,respectively, and 2F4, SEQ ID NOs; 19 and 20 SEQ ID NOs: 17 and 18,respectively. The CDR sequences are in bold.

FIG. 4 shows a FACS analysis which demonstrates that the humanmonoclonal antibodies 7H3, 10C6, 5F1 and 6B4 recognise an epitope onhCMV protein gB.

FIG. 5 shows the nucleotide and amino acid sequences of the heavy andlight chains of 5A2, SEQ ID NOs: 41 and 42 and SEQ ID NOs: 39 and 40,respectively. The CDR sequences are in bold.

The invention is based on the production of antibodies and antibodyfragments that neutralise hCMV infection and which have a particularlyhigh potency in neutralising hCMV infection. Such antibodies aredesirable, as only low concentrations are required in order toneutralise a given amount of virus. This facilitates higher levels ofprotection whilst administering lower amounts of antibody. Humanmonoclonal antibodies and the immortalised B cell clones that secretesuch antibodies are also included within the scope of the invention.

The inventors have discovered that antibodies directed to a combinationof UL130 and UL131A are particularly effective in neutralising hCMV. Thecombination may be a complex of UL130 and UL131A forming an epitoperecognised by the antibody or an antibody may be directed to one ofUL130 and UL131A, the presence of the other protein being necessary forspecificity.

The invention also relates to the characterisation of the epitope towhich the antibodies bind and the use of that epitope in raising animmune response.

The invention also relates to various methods and uses involving theantibodies of the invention and the epitopes to which they bind.

Antibodies

The invention provides monoclonal or recombinant antibodies havingparticularly high potency in neutralising hCMV. The invention alsoprovides fragments of these recombinant or monoclonal antibodies,particularly fragments that retain the antigen-binding activity of theantibodies, for example which retain at least one complementaritydetermining region (CDR) specific for hCMV proteins UL130 and UL131A.

In this specification, by “high potency in neutralising hCMV” is meantthat an antibody molecule of the invention neutralises hCMV in astandard assay at a concentration much lower than antibodies known inthe art, for example compared to MSL-109.

Preferably, the antibody molecule of the present invention canneutralise at a concentration of 0.16 μg/ml or lower (i.e. 0.15, 0.125,0.1, 0.075, 0.05, 0.025, 0.02, 0.016, 0.015, 0.0125, 0.01, 0.0075,0.005, 0.004 or lower), preferably 0.016 μg/ml or lower (an antibodyconcentration of 10⁻⁸ or lower, preferably 10⁻⁹ M or lower, preferably10⁻¹⁰ M or lower, i.e. 10⁻¹¹M, 10⁻¹²M, 10⁻¹³M or lower). This means thatonly very low concentrations of antibody are required for 50%neutralisation of a clinical isolate of hCMV in vitro compared to theconcentration of MSL-109 required for neutralisation of the same titreof hCMV. Preferably, the concentration of antibody of the inventionrequired for 50% neutralisation of infection of endothelial cells,epithelial cells and dendritic cells by a clinical isolate of hCMV is 50times or more (i.e. 75, 100, 150, 200 or more) lower than that requiredby MSL-109. Potency can be measured using a standard neutralisationassay as described in the art.

The antibodies of the invention are able to neutralise hCMV. Preferably,an antibody according to the invention prevents infection of fibroblastsor endothelial cells. More preferably, an antibody according to theinvention prevents infection of endothelial cells. Preferably, anantibody according to the invention prevents infection of bothfibroblasts and endothelial cells. The antibodies of the inventionpreferably also prevent infection of dendritic cells and epithelialcells such as retinal cells.

These antibodies can be used as prophylactic or therapeutic agents uponappropriate formulation, or as a diagnostic tool.

A “neutralising antibody” is one that can neutralise the ability of thatpathogen to initiate and/or perpetuate an infection in a host. Theinvention provides a neutralising monoclonal human antibody, wherein theantibody recognises an antigen from human cytomegalovirus (hCMV).

Preferably, an antibody according to the invention has specificity for acombination of UL130 and UL131A.

Preferably an antibody according to the invention is a monoclonalantibody referred to herein as 1F11 or 2F4. These antibodies wereinitially isolated from a hCMV infected donor, and are produced by theimmortalised B cell clones referred to as 1F11 or 2F4. These antibodieshave been shown to neutralise hCMV infection of endothelial cells,epithelial cells, retinal cells and dendritic cells. In addition, theantibodies 5A2 and 9A11, isolated from a different hCMV infected donor,show the same specificity for a combination of UL130 and UL131A and theability to neutralise hCMV infection of endothelial, epithelial, retinaland dendritic cells. These antibodies are produced by the immortalised Bcell clones referred to as 5A2 and 9A11, respectively.

1F11 consists of a heavy chain having the amino acid sequence recited inSEQ ID NO: 7 and a light chain having the amino acid sequence recited inSEQ ID NO: 8. 2F4 consists of a heavy chain having the amino acidsequence recited in SEQ ID NO: 17 and a light chain having the aminoacid sequence recited in SEQ ID NO: 18. The CDRs of the antibody heavychains are referred to as CDRH1, CDRH2 and CDRH3, respectively.Similarly, the CDRs of the antibody light chains are referred to asCDRL1, CDRL2 and CDRL3, respectively. The position of the CDR aminoacids are defined according to the IMGT numbering system [12, 13, 14]as: CDR1-IMGT positions 27 to 38, CDR2-IMGT positions 56 to 65 andCDR3-IMGT positions 105 to 117.

5A2 consists of a heavy chain having the amino acid sequence recited inSEQ ID NO: 39 and a light chain having the amino acid sequence recitedin SEQ ID NO: 40.

The amino acid sequences of the CDRs of these antibodies are shown inTable 1.

TABLE 1 1F11 2F4 5A2 CDRH1 GFTFSSYA (SEQ ID NO: 1)GFSFNTYG (SEQ ID NO: 11) GGTFSSYV (SEQ ID NO: 33) CDRH2ISFDGDNK (SEQ ID NO: 2) IWDDGSKM (SEQ ID NO: 12)IIPIFNTA (SEQ ID NO: 34) CDRH3 AREELVGLMPPYYNYGLDV ARDEGAIMLHAMTDYGLDVARDFLSGPMEMPGGYYGLDV (SEQ ID NO: 3) (SEQ ID NO: 13) (SEQ ID NO: 35)CDRL1 SSNIGNNF (SEQ ID NO: 4) NLGDEF (SEQ ID NO: 14)QSVLYSSNNKNY (SEQ ID NO: 36) CDRL2 DND (SEQ ID NO:5) QDS (SEQ ID NO: 15)WAS (SEQ ID NO: 37) CDRL3 ETWDGSLNPAVV (SEQ ID QAWDSSTAHYV (SEQ IDQQYYSTPIT (SEQ ID NO: 6) NO: 16) NO: 38)

The invention also includes an antibody comprising a heavy chaincomprising one or more (i.e. one, two or all three) heavy chain CDRsfrom 1F11 or 2F4 (SEQ ID NOs: 1-3 or 11-13). Also included is anantibody comprising a heavy chain comprising one or more (i.e. one, twoor all three) heavy chain CDRs from 5A2 (SEQ ID NOs:33-35).

Preferably an antibody according to the invention comprises a heavychain comprising (i) SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2 andSEQ ID NO: 3 for CDRH3, or (ii) SEQ ID NO: 11 for CDRH1, SEQ ID NO: 12for CDRH2 and SEQ ID NO: 13 for CDRH3. A further preferred antibodyaccording to the invention comprises a heavy chain comprising SEQ ID NO:33 for CDRH1, SEQ ID NO: 34 for CDRH2 and SEQ ID NO: 35 for CDRH3.

The invention also includes an antibody comprising a light chaincomprising one or more (i.e. one, two or all three) light chain CDRsfrom 1F11 or 2F4 (SEQ ID NOs: 4-6 or 14-16). Also included is anantibody comprising a light chain comprising one or more (i.e. one, twoor all three) light chain CDRs from 5A2 (SEQ ID NOs:36-38).

Preferably an antibody according to the invention comprises a lightchain comprising (i) SEQ ID NO: 4 for CDRL1, SEQ ID NO: 5 for CDRL2 andSEQ ID NO: 6 for CDRL3, or (ii) SEQ ID NO: 14 for CDRL1, SEQ ID NO: 15for CDRL2 and SEQ ID NO: 16 for CDRL3. A further preferred antibodyaccording to the invention comprises a light chain comprising SEQ ID NO:36 for CDRL1, SEQ ID NO: 37 for CDRL2 and SEQ ID NO: 38 for CDRL3.

Preferably an antibody according to the invention comprises a heavychain having the sequence recited in SEQ ID NO: 7, 17 or 39. Preferablyan antibody according to the invention comprises a light chain havingthe sequence recited in SEQ ID NO: 8, 18 or 40.

Hybrid antibody molecules may also exist that comprise one or more CDRsfrom 1F11 and one or more CDRs from 2F4. Preferably, such hybridantibodies comprise three CDRs from 1F11 and three CDRs from 2F4. Thus,preferred hybrid antibodies comprise i) the three light chain CDRs from1F11 and the three heavy chain CDRs from 2F4, or ii) the three heavychain CDRs from 1F11 and the three light chain CDRs from 2F4. In analternative, such hybrids may contain one or more CDRs from 5A2.

The invention also includes nucleic acid sequences encoding part or allof the light and heavy chains and CDRs of the present invention.Preferred nucleic acid sequences according to the invention include SEQID NO: 9 (encoding the 1F11 heavy chain variable region), SEQ ID NO: 10(encoding the 1F11 light chain variable region), SEQ ID NO: 19 (encodingthe 2F4 heavy chain variable region), and SEQ ID NO: 20 (encoding the2F4 light chain variable region). Preferred nucleic acid sequencesencoding the various CDRs include SEQ ID NO: 21 (encoding 1F11 CDRH1),SEQ ID NO: 22 (encoding 1F11 CDRH2), SEQ ID NO:23 (encoding 1F11 CDRH3),SEQ ID NO:24 (encoding 1F11 CDRL1), SEQ ID NO:25 (encoding 1F11 CDRL2),SEQ ID NO:26 (encoding 1F11 CDRL3), SEQ ID NO:27 (encoding 2F4 CDRH1),SEQ ID NO:28 (encoding 2F4 CDRH2), SEQ ID NO:29 (encoding 2F4 CDRH3),SEQ ID NO:30 (encoding 2F4 CDRL1), SEQ ID NO:31 (encoding 2F4 CDRL2) andSEQ ID NO:32 (encoding 2F4 CDRL3). Further preferred nucleic acidsequences according to the invention include SEQ ID NO: 41 (encoding the5A2 heavy chain variable region), SEQ ID NO: 42 (encoding the 5A2 lightchain variable region), SEQ ID NO: 43 (encoding 5A2 CDRH1), SEQ ID NO:44 (encoding 5A2 CDRH2), SEQ ID NO:45 (encoding 5A2 CDRH3), SEQ ID NO:46(encoding 5A2 CDRL1), SEQ ID NO:47 (encoding 5A2 CDRL2), SEQ ID NO:48(encoding 5A2 CDRL3). Due to the redundancy of the genetic code,variants of these sequences will exist that encode the same amino acidsequences.

Variant antibodies are also included within the scope of the invention.Thus, variants of the sequences recited in the application are alsoincluded within the scope of the invention. Such variants may arise dueto the degeneracy of the genetic code, as mentioned above.Alternatively, natural variants may be produced due to errors intranscription or translation. A variant of 2F4 is also disclosed herein.This variant comprises an additional two serine residues at theC-terminal end of the 2F4 heavy chain amino acid sequence (SEQ ID NO:17). Thus, this variant of 2F4 consists of a heavy chain having theamino acid sequence recited in SEQ ID NO:49 and a light chain having theamino acid sequence recited in SEQ ID NO: 18. The nucleic acid sequenceencoding the variant heavy chain is recited in SEQ ID NO:50. Thus,antibodies comprising the 2F4 variant heavy chain (SEQ ID NO: 49) areincluded within the scope of the invention.

Further variants of the antibody sequences having improved affinity maybe obtained using methods known in the art and are included within thescope of the invention. For example, amino acid substitutions may beused to obtain antibodies with further improved affinity. Alternatively,codon optimisation of the nucleotide sequence may be used to improve theefficiency of translation in expression systems for the production ofthe antibody.

Preferably, such variant antibody sequences will share 70% or more (i.e.80, 85, 90, 95, 97, 98, 99% or more) sequence identity with thesequences recited in the application. Preferably such sequence identityis calculated with regard to the full length of the reference sequence(i.e. the sequence recited in the application). Preferably, percentageidentity, as referred to herein, is as determined using BLAST version2.1.3 using the default parameters specified by the NCBI (the NationalCenter for Biotechnology Information) [Blosum 62 matrix; gap openpenalty=11 and gap extension penalty=1].

Further included within the scope of the invention are vectors, forexample expression vectors, comprising a nucleic acid sequence accordingto the invention. Cells transformed with such vectors are also includedwithin the scope of the invention.

The invention also relates to monoclonal antibodies that bind to anepitope capable of binding the monoclonal antibody 1F11 or 2F4. Theinvention also relates to monoclonal antibodies that bind to an epitopecapable of binding the monoclonal antibody 5A2.

Monoclonal and recombinant antibodies are particularly useful inidentification and purification of the individual polypeptides or otherantigens against which they are directed. The antibodies of theinvention have additional utility in that they may be employed asreagents in inmiunoassays, radioimmunoassays (RIA) or enzyme-linkedimmunosorbent assays (ELISA). In these applications, the antibodies canbe labelled with an analytically-detectable reagent such as aradioisotope, a fluorescent molecule or an enzyme. The antibodies mayalso be used for the molecular identification and characterisation(epitope mapping) of antigens.

Antibodies of the invention can be coupled to a drug for delivery to atreatment site or coupled to a detectable label to facilitate imaging ofa site comprising cells of interest, such as cells infected with hCMV.Methods for coupling antibodies to drugs and detectable labels are wellknown in the art, as are methods for imaging using detectable labels.Labeled antibodies may be employed in a wide variety of assays,employing a wide variety of labels. Detection of the formation of anantibody-antigen complex between an antibody of the invention and anepitope of interest (an hCMV epitope) can be facilitated by attaching adetectable substance to the antibody. Suitable detection means includethe use of labels such as radionuclides, enzymes, coenzymes,fluorescers, chemiluminescers, chromogens, enzyme substrates orco-factors, enzyme inhibitors, prosthetic group complexes, freeradicals, particles, dyes, and the like. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase, β-galactosidase,or acetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material isluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S, or ³H. Such labeled reagents may be used in avariety of well-known assays, such as radioimmunoassays, enzymeimmunoassays, e.g., ELISA, fluorescent immunoassays, and the like. Seefor example, references 15-18.

An antibody according to the invention may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent, or aradioactive metal ion or radioisotope. Examples of radioisotopesinclude, but are not limited to, I-131, I-123, I-125, Y-90, Re-188,Re-186, At-211, Cu-67, Bi-212, Bi-213, Pd-109, Tc-99, In-111, and thelike. Such antibody conjugates can be used for modifying a givenbiological response; the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin.

Techniques for conjugating such therapeutic moiety to antibodies arewell known. See, for example, Arnon et al. (1985) “Monoclonal Antibodiesfor Immunotargeting of Drugs in Cancer Therapy,” in MonoclonalAntibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.),pp. 243-256; ed. Hellstrom et al. (1987) “Antibodies for Drug Delivery,”in Controlled Drug Delivery, ed. Robinson et al. (2d ed; Marcel Dekker,Inc.), pp. 623-653; Thorpe (1985) “Antibody Carriers of Cytotoxic Agentsin Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biologicaland Clinical Applications, ed. Pinchera et al. pp. 475-506 (EditriceKurtis, Milano, Italy, 1985); “Analysis, Results, and Future Prospectiveof the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy,” inMonoclonal Antibodies for Cancer Detection and Therapy, ed. Baldwin etal. (Academic Press, New York, 1985), pp. 303-316; and Thorpe et al.(1982) Immunol. Rev. 62:119-158.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described in reference 19. Inaddition, linkers may be used between the labels and the antibodies ofthe invention [20]. Antibodies or, antigen-binding fragments thereof maybe directly labeled with radioactive iodine, indium, yttrium, or otherradioactive particle known in the art [21]. Treatment may consist of acombination of treatment with conjugated and nonconjugated antibodiesadministered simultaneously or subsequently [22, 23].

Antibodies of the invention may be attached to a solid support.

Additionally, antibodies can be chemically modified by covalentconjugation to a polymer to increase their circulating half-life, forexample. Preferred polymers, and methods to attach them to peptides, areshown in references 24-27. Preferred polymers are polyoxyethylatedpolyols and polyethylene glycol (PEG). PEG is soluble in water at roomtemperature and has the general formula: R(O—CH₂—CH₂)_(n)O—R where R canbe hydrogen, or a protective group such as an alkyl or alkanol group.Preferably, the protective group has between 1 and 8 carbons, morepreferably it is methyl. The symbol n is a positive integer, preferablybetween 1 and 1,000, more preferably between 2 and 500. The PEG has apreferred average molecular weight between 1,000 and 40,000, morepreferably between 2,000 and 20,000, most preferably between 3,000 and12,000. Preferably, PEG has at least one hydroxy group, more preferablyit is a terminal hydroxy group. It is this hydroxy group which ispreferably activated to react with a free amino group on the inhibitor.

However, it will be understood that the type and amount of the reactivegroups may be varied to achieve a covalently conjugated PEG/antibody ofthe present invention.

Water-soluble polyoxyethylated polyols are also useful in the presentinvention. They include polyoxyethylated sorbitol, polyoxyethylatedglucose, polyoxyethylated glycerol (POG), and the like. POG ispreferred. One reason is because the glycerol backbone ofpolyoxyethylated glycerol is the same backbone occurring naturally in,for example, animals and humans in mono-, di-, triglycerides. Therefore,this branching would not necessarily be seen as a foreign agent in thebody. The POG has a preferred molecular weight in the same range as PEG.The structure for POG is shown in reference 28, and a discussion ofPOG/IL-2 conjugates is found in reference 24.

Another drug delivery system for increasing circulatory half-life is theliposome. Methods of preparing liposome delivery systems are discussedin references 29, 30 and 31. Other drug delivery systems are known inthe art and are described in, for example, references 32 and 33.

Antibodies of the invention are preferably provided in purified form.Typically, the antibody will be present in a composition that issubstantially free of other polypeptides e.g. where less than 90% (byweight), usually less than 60% and more usually less than 50% of thecomposition is made up of other polypeptides.

Antibodies of the invention may be immunogenic in non-human (orheterologous) hosts e.g. in mice. In particular, the antibodies may havean idiotope that is immunogenic in non-human hosts, but not in a humanhost. Antibodies of the invention for human use include those thatcannot be obtained from hosts such as mice, goats, rabbits, rats,non-primate mammals, etc. and cannot be obtained by humanisation or fromxeno-mice.

Antibodies of the invention can be of any isotype (e.g. IgA, IgG, IgMi.e. an α, γ or μ heavy chain), but will generally be IgG. Within theIgG isotype, antibodies may be IgG1, IgG2, IgG3 or IgG4 subclass.Antibodies of the invention may have a κ or a λ light chain.

Production of Antibodies

Monoclonal antibodies according to the invention can be made by one ofthe methods known in the art. The general methodology for makingmonoclonal antibodies using hybridoma technology is well known [34, 35].Preferably, the alternative EBV immortalisation method described inreference 36 is used.

Using the method described in reference 36, B-cells producing theantibody of the invention can be transformed with EBV in the presence ofa polyclonal B cell activator. Transformation with EBV is a standardtechnique and can easily be adapted to include polyclonal B cellactivators.

Additional stimulants of cellular growth and differentiation may beadded during the transformation step to further enhance the efficiency.These stimulants may be cytokines such as IL-2 and IL-15. In aparticularly preferred aspect, IL-2 is added during the immortalisationstep to further improve the efficiency of immortalisation, but its useis not essential.

The immortalised B cells produced using these methods can then becultured using methods known in the art and antibodies isolatedtherefrom.

Monoclonal antibodies may be further purified, if desired, usingfiltration, centrifugation and various chromatographic methods such asHPLC or affinity chromatography. Techniques for purification ofmonoclonal antibodies, including techniques for producingpharmaceutical-grade antibodies, are well known in the art.

Fragments of the monoclonal antibodies of the invention can be obtainedfrom the monoclonal antibodies by methods that include digestion withenzymes, such as pepsin or papain, and/or by cleavage of disulfide bondsby chemical reduction. Alternatively, fragments of the monoclonalantibodies can be obtained by cloning and expression of part ofsequences of the heavy or light chains. Antibody “fragments” includeFab, Fab′, F(ab′)₂ and Fv fragments. The invention also encompassessingle-chain Fv fragments (scFv) derived from the heavy and light chainsof a monoclonal antibody of the invention e.g. the invention includes ascFv comprising the CDRs from an antibody of the invention. Alsoincluded are heavy or light chain monomers and dimers as well as singlechain antibodies, e.g. single chain Fv in which the heavy and lightchain variable domains are joined by a peptide linker

Standard techniques of molecular biology may be used to prepare DNAsequences coding for the antibodies or fragments of the antibodies ofthe present invention. Desired DNA sequences may be synthesisedcompletely or in part using oligonucleotide synthesis techniques.Site-directed mutagenesis and polymerase chain reaction (PCR) techniquesmay be used as appropriate.

Any suitable host cell/vector system may be used for expression of theDNA sequences encoding the antibody molecules of the present inventionor fragments thereof. Bacterial, for example E. coli, and othermicrobial systems may be used, in part, for expression of antibodyfragments such as Fab and F(ab′)₂ fragments, and especially Fv fragmentsand single chain antibody fragments, for example, single chain Fvs.Eukaryotic, e g mammalian, host cell expression systems may be used forproduction of larger antibody molecules, including complete antibodymolecules. Suitable mammalian host cells include CHO, HEK293T, PER.C6,myeloma or hybridoma cells.

The present invention also provides a process for the production of anantibody molecule according to the present invention comprisingculturing a host cell comprising a vector of the present invention underconditions suitable for leading to expression of protein from DNAencoding the antibody molecule of the present invention, and isolatingthe antibody molecule.

The antibody molecule may comprise only a heavy or light chainpolypeptide, in which case only a heavy chain or light chain polypeptidecoding sequence needs to be used to transfect the host cells. Forproduction of products comprising both heavy and light chains, the cellline may be transfected with two vectors, a first vector encoding alight chain polypeptide and a second vector encoding a heavy chainpolypeptide. Alternatively, a single vector may be used, the vectorincluding sequences encoding light chain and heavy chain polypeptides.

Alternatively, antibodies according to the invention may be produced byi) expressing a nucleic acid sequence according to the invention in acell, and ii) isolating the expressed antibody product. Additionally,the method may include iii) purifying the antibody.

Screening and Isolation of B Cells

Transformed B cells are screened for those producing antibodies of thedesired antigen specificity, and individual B cell clones can then beproduced from the positive cells.

The screening step may be carried out by ELISA, by staining of tissuesor cells (including transfected cells), a neutralisation assay or one ofa number of other methods known in the art for identifying desiredantigen specificity. The assay may select on the basis of simple antigenrecognition, or may select on the additional basis of a desired functione.g. to select neutralising antibodies rather than just antigen-bindingantibodies, to select antibodies that can change characteristics oftargeted cells, such as their signalling cascades, their shape, theirgrowth rate, their capability of influencing other cells, their responseto the influence by other cells or by other reagents or by a change inconditions, their differentiation status, etc.

The cloning step for separating individual clones from the mixture ofpositive cells may be carried out using limiting dilution,micromanipulation, single cell deposition by cell sorting or anothermethod known in the art. Preferably the cloning is carried out usinglimiting dilution.

The immortalised B cell clones of the invention can be used in variousways e.g. as a source of monoclonal antibodies, as a source of nucleicacid (DNA or mRNA) encoding a monoclonal antibody of interest, forresearch, etc.

The invention provides a composition comprising immortalised B memorylymphocytes, wherein the lymphocytes produce antibodies with highneutralising potency specific for hCMV, and wherein the antibodies areproduced at ≧5 pg per cell per day. The invention also provides acomposition comprising clones of an immortalised B memory lymphocyte,wherein the clones produce a monoclonal antibody with a high affinityspecific for hCMV, and wherein the antibody is produced at ≧10^(N) ngper clone per day. Preferably said clones produce a monoclonal antibodywith a high potency in neutralizing hCMV infection.

Preferred immortalised B cell clones according to the invention are 1F11and 2F4. Further preferred clones are 5A2 and 9A11.

Epitopes

As mentioned above, the antibodies of the invention can be used to mapthe epitopes to which they bind. The Applicants have discovered that theantibodies 1F11, 2F4, 5A2 and 9A11 that neutralise hCMV infection ofendothelial cells, epithelial cells, retinal cells and dendritic cellsare directed towards an epitope found on a combination of UL130 andUL131A. Although the Applicant does not wish to be bound by this theory,it is believed that the antibodies 1F11 and 2F4 bind to a conformationalepitope formed by these two proteins. It is also believed that 5A2 and9A11 also bind to such a conformational epitope formed by UL130 andUL131A.

Due to the fact that 1F11, 2F4, 5A2, and 9A11 do not neutraliseinfection of fibroblasts, it is postulated that these antibodiesrecognise a different epitope to the human monoclonal antibodies 1006,5F1, 6B4 and 7H3. Furthermore, it is believed that the monoclonalantibodies 1006, 5F1, 7H3 and 6B4 bind to a functional epitope of gB.

The epitopes recognised by these antibodies may have a number of uses.The epitopes and mimotopes in purified or synthetic form can be used toraise immune responses (i.e., as a vaccine, or for the production ofantibodies for other uses) or for screening patient serum for antibodiesthat immunoreact with the epitopes or mimotopes. Preferably, such anepitope or mimotope, or antigen comprising such an epitope or mimotopeis used as a vaccine for raising an immune response. The antibodies ofthe invention can also be used in a method to monitor the quality ofvaccines in particular to check that the antigen in a vaccine containsthe correct immunogenic epitope in the correct conformation.

The epitopes may also be useful in screening for ligands that bind tosaid epitopes. Such ligands preferably block the epitopes and so preventinfection. Such ligands are encompassed within the scope of theinvention.

Recombinant Expression

The immortalised memory B lymphocytes of the invention may also be usedas a source of nucleic acid for the cloning of antibody genes forsubsequent recombinant expression. Expression from recombinant sourcesis more common for pharmaceutical purposes than expression from B cellsor hybridomas e.g. for reasons of stability, reproducibility, cultureease, etc.

Thus the invention provides a method for preparing a recombinant cell,comprising the steps of: (i) obtaining one or more nucleic acids (e.g.heavy and/or light chain genes) from the B cell clone that encodes theantibody of interest; and (ii) inserting the nucleic acid into anexpression host in order to permit expression of the antibody ofinterest in that host.

Similarly, the invention provides a method for preparing a recombinantcell, comprising the steps of: (i) sequencing nucleic acid(s) from the Bcell clone that encodes the antibody of interest; and (ii) using thesequence information from step (i) to prepare nucleic acid(s) forinserting into an expression host in order to permit expression of theantibody of interest in that host.

The invention also provides a method of preparing a recombinant cell,comprising the step of transforming a host cell with one or more nucleicacids that encode a monoclonal antibody of interest, wherein the nucleicacids are nucleic acids that were derived from an immortalised B cellclone of the invention. Thus the procedures for first preparing thenucleic acid(s) and then using it to transform a host cell can beperformed at different times by different people in different places(e.g. in different countries).

These recombinant cells of the invention can then be used for expressionand culture purposes. They are particularly useful for expression ofantibodies for large-scale pharmaceutical production. They can also beused as the active ingredient of a pharmaceutical composition. Anysuitable culture techniques can be used, including but not limited tostatic culture, roller bottle culture, ascites fluid, hollow-fiber typebioreactor cartridge, modular minifermenter, stirred tank, microcarrierculture, ceramic core perfusion, etc.

Methods for obtaining and sequencing immunoglobulin genes from B cellsare well known in the art e.g. see reference 37).

The expression host is preferably a eukaryotic cell, including yeast andanimal cells, particularly mammalian cells (e.g. CHO cells, human cellssuch as PER.C6 [Crucell; reference 38] or HKB-11 [Bayer; references 39 &40] cells, myeloma cells [41 & 42], etc.), as well as plant cells.Preferred expression hosts can glycosylate the antibody of theinvention, particularly with carbohydrate structures that are notthemselves immunogenic in humans. Expression hosts that can grow inserum-free media are preferred. Expression hosts that can grow inculture without the presence of animal-derived products are preferred.

The expression host may be cultured to give a cell line.

The invention provides a method for preparing one or more nucleic acidmolecules (e.g. heavy and light chain genes) that encodes an antibody ofinterest, comprising the steps of: (i) preparing an immortalised B cellclone according to the invention; (ii) obtaining from the B cell clonenucleic acid that encodes the antibody of interest. The invention alsoprovides a method for obtaining a nucleic acid sequence that encodes anantibody of interest, comprising the steps of: (i) preparing animmortalised B cell clone according to the invention; (ii) sequencingnucleic acid from the B cell clone that encodes the antibody ofinterest.

The invention also provides a method of preparing nucleic acidmolecule(s) that encodes an antibody of interest, comprising the step ofobtaining the nucleic acid from a B cell clone that was obtained from atransformed B cell of the invention. Thus the procedures for firstobtaining the B cell clone and then preparing nucleic acid(s) from itcan be performed at very different times by different people indifferent places (e.g. in different countries).

The invention provides a method for preparing an antibody (e.g. forpharmaceutical use), comprising the steps of: (i) obtaining and/orsequencing one or more nucleic acids (e.g. heavy and light chain genes)from the selected B cell clone expressing the antibody of interest; (ii)inserting the nucleic acid(s) into or using the nucleic acid(s) toprepare an expression host that can express the antibody of interest;(iii) culturing or sub-culturing the expression host under conditionswhere the antibody of interest is expressed; and, optionally, (iv)purifying the antibody of the interest.

The invention also provides a method of preparing an antibody comprisingthe steps of: culturing or sub-culturing an expression host cellpopulation under conditions where the antibody of interest is expressedand, optionally, purifying the antibody of the interest, wherein saidexpression host cell population has been prepared by (i) providingnucleic acid(s) encoding a selected B cell the antibody of interest thatis produced by a population of B memory lymphocytes prepared asdescribed above, (ii) inserting the nucleic acid(s) into an expressionhost that can express the antibody of interest, and (iii) culturing orsub-culturing expression hosts comprising said inserted nucleic acids toproduce said expression host cell population. Thus the procedures forfirst preparing the recombinant expression host and then culturing it toexpress antibody can be performed at very different times by differentpeople in different places (e.g. in different countries).

Pharmaceutical Compositions

The use of antibodies as the active ingredient of pharmaceuticals is nowwidespread, including the products Herceptin™ (trastuzumab), Rituxan™,Campath™, Remicade™, ReoPro™, Mylotarg™, Zevalin™, Omalizumab, Synagis™(Palivizumab), Zenapax™ (daclizumab), etc.

The invention thus provides a pharmaceutical composition containing theantibodies of the invention and/or nucleic acid encoding such antibodiesand/or immortalised B cells that express such antibodies and/or theepitopes recognised by the antibodies of the invention. A pharmaceuticalcomposition may also contain a pharmaceutically acceptable carrier toallow administration. The carrier should not itself induce theproduction of antibodies harmful to the individual receiving thecomposition and should not be toxic. Suitable carriers may be large,slowly metabolised macromolecules such as proteins, polypeptides,liposomes, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers and inactive virusparticles.

Pharmaceutically acceptable salts can be used, for example mineral acidsalts, such as hydrochlorides, hydrobromides, phosphates and sulphates,or salts of organic acids, such as acetates, propionates, malonates andbenzoates.

Pharmaceutically acceptable carriers in therapeutic compositions mayadditionally contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents or pH buffering substances, may be present in suchcompositions. Such carriers enable the pharmaceutical compositions to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries and suspensions, for ingestion by the patient.

Preferred forms for administration include forms suitable for parenteraladministration, e.g. by injection or infusion, for example by bolusinjection or continuous infusion. Where the product is for injection orinfusion, it may take the form of a suspension, solution or emulsion inan oily or aqueous vehicle and it may contain formulatory agents, suchas suspending, preservative, stabilising and/or dispersing agents.Alternatively, the antibody molecule may be in dry form, forreconstitution before use with an appropriate sterile liquid.

Once formulated, the compositions of the invention can be administereddirectly to the subject. It is preferred that the compositions areadapted for administration to human subjects.

The pharmaceutical compositions of this invention may be administered byany number of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intraperitoneal,intrathecal, intraventricular, transdermal, transcutaneous, topical,subcutaneous, intranasal, enteral, sublingual, intravaginal or rectalroutes. Hyposprays may also be used to administer the pharmaceuticalcompositions of the invention. Typically, the therapeutic compositionsmay be prepared as injectables, either as liquid solutions orsuspensions. Solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection may also be prepared.

Direct delivery of the compositions will generally be accomplished byinjection, subcutaneously, intraperitoneally, intravenously orintramuscularly, or delivered to the interstitial space of a tissue. Thecompositions can also be administered into a lesion. Dosage treatmentmay be a single dose schedule or a multiple dose schedule. Knownantibody-based pharmaceuticals provide guidance relating to frequency ofadministration e.g. whether a pharmaceutical should be delivered daily,weekly, monthly, etc. Frequency and dosage may also depend on theseverity of symptoms.

Compositions of the invention may be prepared in various forms. Forexample, the compositions may be prepared as injectables, either asliquid solutions or suspensions. Solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection can also beprepared (e.g. a lyophilised composition, like Synagis™ and Herceptin™,for reconstitution with sterile water containing a preservative). Thecomposition may be prepared for topical administration e.g. as anointment, cream or powder. The composition may be prepared for oraladministration e.g. as a tablet or capsule, as a spray, or as a syrup(optionally flavoured). The composition may be prepared for pulmonaryadministration e.g. as an inhaler, using a fine powder or a spray. Thecomposition may be prepared as a suppository or pessary. The compositionmay be prepared for nasal, aural or ocular administration e.g. as drops.The composition may be in kit form, designed such that a combinedcomposition is reconstituted just prior to administration to a patient.For example, a lyophilised antibody can be provided in kit form withsterile water or a sterile buffer.

It will be appreciated that the active ingredient in the compositionwill be an antibody molecule. As such, it will be susceptible todegradation in the gastrointestinal tract. Thus, if the composition isto be administered by a route using the gastrointestinal tract, thecomposition will need to contain agents which protect the antibody fromdegradation but which release the antibody once it has been absorbedfrom the gastrointestinal tract.

A thorough discussion of pharmaceutically acceptable carriers isavailable in Gennaro (2000) Remington: The Science and Practice ofPharmacy, 20th edition, ISBN: 0683306472.

Pharmaceutical compositions of the invention generally have a pH between5.5 and 8.5, preferably between 6 and 8, and more preferably about 7.The pH may be maintained by the use of a buffer. The composition may besterile and/or pyrogen free. The composition may be isotonic withrespect to humans Pharmaceutical compositions of the invention arepreferably supplied in hermetically-sealed containers.

Pharmaceutical compositions will include an effective amount of one ormore antibodies of the invention and/or one or more immortalised B cellsof the invention and/or a polypeptide comprising an epitope that bindsan antibody of the invention i.e. an amount that is sufficient to treat,ameliorate, or prevent a desired disease or condition, or to exhibit adetectable therapeutic effect. Therapeutic effects also includereduction in physical symptoms. The precise effective amount for anyparticular subject will depend upon their size and health, the natureand extent of the condition, and the therapeutics or combination oftherapeutics selected for administration. The effective amount for agiven situation is determined by routine experimentation and is withinthe judgment of a clinician. For purposes of the present invention, aneffective dose will generally be from about 0.01 mg/kg to about 50mg/kg, or about 0.05 mg/kg to about 10 mg/kg of the compositions of thepresent invention in the individual to which it is administered. Knownantibody-based pharmaceuticals provide guidance in this respect e.g.Herceptin™ is administered by intravenous infusion of a 21 mg/mlsolution, with an initial loading dose of 4 mg/kg body weight and aweekly maintenance dose of 2 mg/kg body weight; Rituxan™ is administeredweekly at 375 mg/m²; etc.

Compositions can include more than one (e.g. 2, 3, 4, 5, etc.) antibodyof the invention, particularly where such antibodies bind to differentantigens (or to different epitopes in the same antigen) to provide anadditive or synergistic therapeutic effect. For example, one antibodymay bind to the UL130-UL131A combination (or complex) while another maybind to gH. In a further example, one antibody may bind to theUL130-UL131A combination (or complex) while another may bind to gB.Thus, one antibody may be targeted to the mechanism that mediatesinfection of fibroblasts, while the other antibody may be targeted tothe mechanism that mediates infection of endothelial cells. For optimalclinical effect it may well be advantageous to address both mechanismsof hCMV infection and maintenance.

Antibodies of the invention may be administered (either combined orseparately) with other therapeutics e.g. with chemotherapeuticcompounds, with radiotherapy, etc. Preferred therapeutic compoundsinclude anti-viral compounds such as ganciclovir, foscarnet andcidofovir. Such combination therapy provides an additive or synergisticimprovement in therapeutic efficacy relative to the individualtherapeutic agents when administered alone. The term “synergy” is usedto describe a combined effect of two or more active agents that isgreater than the sum of the individual effects of each respective activeagent. Thus, where the combined effect of two or more agents results in“synergistic inhibition” of an activity or process, it is intended thatthe inhibition of the activity or process is greater than the sum of theinhibitory effects of each respective active agent. The term“synergistic therapeutic effect” refers to a therapeutic effect observedwith a combination of two or more therapies wherein the therapeuticeffect (as measured by any of a number of parameters) is greater thanthe sum of the individual therapeutic effects observed with therespective individual therapies.

Antibodies may be administered to those patients who have previouslyshown no response to treatment for hCMV infection, i.e. have been shownto be refractive to anti-hCMV treatment. Such treatment may includeprevious treatment with an anti-viral agent. This may be due to, forexample, infection with an anti-viral resistant strain of hCMV.

In compositions of the invention that include antibodies of theinvention, the antibodies preferably make up at least 50% by weight(e.g. 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more) of the totalprotein in the composition. The antibodies are thus in purified form.

The invention provides a method of preparing a pharmaceutical,comprising the steps of: (i) preparing an antibody of the invention; and(ii) admixing the purified antibody with one or morepharmaceutically-acceptable carriers.

The invention also provides a method of preparing a pharmaceutical,comprising the step of admixing an antibody with one or morepharmaceutically-acceptable carriers, wherein the antibody is amonoclonal antibody that was obtained from a transformed B cell of theinvention. Thus the procedures for first obtaining the monoclonalantibody and then preparing the pharmaceutical can be performed at verydifferent times by different people in different places (e.g. indifferent countries).

As an alternative to delivering antibodies or B cells for therapeuticpurposes, it is possible to deliver nucleic acid (typically DNA) to asubject that encodes the monoclonal antibody (or active fragmentthereof) of interest, such that the nucleic acid can be expressed in thesubject in situ to provide a desired therapeutic effect. Suitable genetherapy and nucleic acid delivery vectors are known in the art.

Compositions of the invention may be immunogenic compositions, and aremore preferably vaccine compositions comprising an antigen comprising anepitope found on a combination of hCMV proteins UL130 and UL131A.Alternative compositions may comprise (i) an antigen comprising anepitope found on a combination of hCMV proteins UL130 and UL131A, and(ii) an antigen comprising an epitope found on hCMV gB. Vaccinesaccording to the invention may either be prophylactic (i.e. to preventinfection) or therapeutic (i.e. to treat infection), but will typicallybe prophylactic.

Compositions may include an antimicrobial, particularly if packaged in amultiple dose format.

Compositions may comprise detergent e.g. a Tween (polysorbate), such asTween 80. Detergents are generally present at low levels e.g. <0.01%.

Compositions may include sodium salts (e.g. sodium chloride) to givetonicity. A concentration of 10±2 mg/ml NaCl is typical.

Compositions may comprise a sugar alcohol (e.g. mannitol) or adisaccharide (e.g. sucrose or trehalose) e.g. at around 15-30 mg/ml(e.g. 25 mg/ml), particularly if they are to be lyophilised or if theyinclude material which has been reconstituted from lyophilised material.The pH of a composition for lyophilisation may be adjusted to around 6.1prior to lyophilisation.

The compositions of the invention may also comprise one or moreimmunoregulatory agents. Preferably, one or more of the immunoregulatoryagents include(s) an adjuvant.

The compositions of the invention will preferably elicit both a cellmediated immune response as well as a humoral immune response in orderto effectively address an hCMV infection. This immune response willpreferably induce long lasting (e.g. neutralising) antibodies and a cellmediated immunity that can quickly respond upon exposure to hCMV.

Medical Treatments and Uses

The antibodies of the invention or fragments thereof may be used for thetreatment of hCMV infection, for the prevention of hCMV infection or forthe diagnosis of hCMV infection.

Methods of diagnosis may include contacting an antibody or an antibodyfragment with a sample. Such samples may be tissue samples taken from,for example, salivary glands, lung, liver, pancrease, kidney, ear, eye,placenta, alimentary tract, heart, ovaries, pituitary, adrenals,thyroid, brain or skin. The methods of diagnosis may also include thedetection of an antigen/antibody complex.

The invention therefore provides (i) an antibody according to theinvention, (ii) an immortalised B cell clone according to the invention,(iii) an epitope capable of binding one of 1F11 or 2F4, or (iv) anepitope capable of binding one of 5A2 or 9A11, for use in therapy.

Also provided is a method of treating a patient comprising administeringto that patient (i) an antibody according to the invention, (ii) anepitope capable of binding one of 1F11 or 2F4, or (iii) an epitopecapable of binding one of 5A2 or 9A11.

The invention also provides the use of (i) an antibody according to theinvention, (ii) an immortalised B cell clone according to the invention,(iii) an epitope capable of binding one of 1F11 or 2F4, (iv) an antibodythat binds to an epitope capable of binding one of 1F11 or 2F4, (v) anepitope capable of binding one of 5A2 or 9A11, or (vi) an antibody thatbinds to an epitope capable of binding one of 5A2 or 9A11, in themanufacture of a medicament for the prevention or treatment of hCMVinfection.

The invention provides a composition of the invention for use as amedicament. It also provides the use of an antibody of the inventionand/or a protein comprising an epitope to which such an antibody bindsin the manufacture of a medicament for treatment of a patient and/ordiagnosis in a patient. It also provides a method for treating a subjectand/or of performing diagnosis on a subject, comprising the step ofadministering to them a composition of the invention. The subject ispreferably a human. One way of checking efficacy of therapeutictreatment involves monitoring disease symptoms after administration ofthe composition of the invention. Treatment can be a single doseschedule or a multiple dose schedule. The invention is useful for CMVinfection.

Preferably, an antibody, immortalised B cell clone, epitope orcomposition according to the invention is administered to groups ofsubjects particularly at risk of or susceptible to hCMV infection. Suchsubjects groups include immunocompromised subjects, such as thosesuffering from HIV or undergoing immunosuppressive therapy, such astransplant patients.

Antibodies of the invention can be used in passive immunisation.

Antibodies and fragments thereof as described in the present inventionmay also be used in a kit for the diagnosis of hCMV infection.

Epitopes capable of binding the monoclonal antibody 1F11 or 2F4described in the present invention may be used in a kit for monitoringthe efficacy of vaccination procedures by detecting the presence ofprotective anti-hCMV antibodies.

Epitopes capable of binding the monoclonal antibody 5A2 or 9A11described in the present invention may be used in a kit for monitoringthe efficacy of vaccination procedures by detecting the presence ofprotective anti-hCMV antibodies.

Antibodies and fragments thereof as described in the present inventionmay also be used in a kit for monitoring vaccine manufacture with thedesired immunogenicity.

The invention also provides a method of preparing a pharmaceutical,comprising the step of admixing a monoclonal antibody with one or morepharmaceutically-acceptable carriers, wherein the monoclonal antibody isa monoclonal antibody that was obtained from an expression host of theinvention. Thus the procedures for first obtaining the monoclonalantibody (e.g. expressing it and/or purifying it) and then admixing itwith the pharmaceutical carrier(s) can be performed at very differenttimes by different people in different places (e.g. in differentcountries).

Starting with a transformed B cell of the invention, various steps ofculturing, sub-culturing, cloning, sub-cloning, sequencing, nucleic acidpreparation etc. can be performed in order to perpetuate the antibodyexpressed by the transformed B cell, with optional optimisation at eachstep. In a preferred embodiment, the above methods further comprisetechniques of optimisation (e.g. affinity maturation or optimisation)applied to the nucleic acids encoding the antibody. The inventionencompasses all cells, nucleic acids, vectors, sequences, antibodiesetc. used and prepared during such steps.

In all these methods, the nucleic acid used in the expression host maybe manipulated between steps (ii) and (iii) to insert, delete or amendcertain nucleic acid sequences. Changes from such manipulation include,but are not limited to, changes to introduce restriction sites, to amendcodon usage, to add or optimise transcription and/or translationregulatory sequences, etc. It is also possible to change the nucleicacid to alter the encoded amino acids. For example, it may be useful tointroduce one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) aminoacid substitutions, one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,etc.) amino acid deletions and/or one or more (e.g. 1, 2, 3, 4, 5, 6, 7,8, 9, 10, etc.) amino acid insertions into the antibody's amino acidsequence. Such point mutations can modify effector functions,antigen-binding affinity, post-translational modifications,immunogenicity, etc., can introduce amino acids for the attachment ofcovalent groups (e.g. labels) or can introduce tags (e.g. forpurification purposes). Mutations can be introduced in specific sites orcan be introduced at random, followed by selection (e.g. molecularevolution).

General

The term “comprising” encompasses “including” as well as “consisting of”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y.

The term “about” in relation to a numerical value x means, for example,x±10%.

The term “disease” as used herein is intended to be generallysynonymous, and is used interchangeably with, the terms “disorder” and“condition” (as in medical condition), in that all reflect an abnormalcondition of the human or animal body or of one of its parts thatimpairs normal functioning, is typically manifested by distinguishingsigns and symptoms, and causes the human or animal to have a reducedduration or quality of life.

As used herein, reference to “treatment” of a patient is intended toinclude prevention and prophylaxis. The term “patient” means all mammalsincluding humans Examples of patients include humans, cows, dogs, cats,horses, goats, sheep, pigs, and rabbits. Preferably, the patient is ahuman.

The following are methods which can be used to practice the invention.

EXAMPLE 1 Cloning of B Cells and Screening for hCMV NeutralisingActivity

Two donors with high hCMV neutralising antibody titres in the serum wereidentified. Memory B cells were isolated and immortalised using EBV andCpG as described in reference 36. Briefly, memory B cells were isolatedby negative selection using CD22 beads, followed by removal of IgM⁺,IgD⁺ IgA⁺ B cells using specific antibodies and cell sorting. The sortedcells (IgG⁺) were immortalised with EBV in the presence of CpG 2006 andirradiated allogeneic mononuclear cells. Replicate cultures eachcontaining 50 memory B cells were set up in twenty 96 well U bottomplates. After two weeks the culture supernatants were collected andtested for their capacity to neutralise hCMV infection of eitherfibroblasts or epithelial cells in separate assays. B cell clones wereisolated from positive polyclonal cultures as described in reference 36.IgG concentrations in the supernatant of selected clones were determinedusing an IgG-specific ELISA.

For the viral neutralisation assay a titrated amount of a clinical hCMVisolate was mixed with an equal volume of culture supernatant or withdilutions of human sera containing neutralising antibodies. After 1 hourincubation at room temperature the mixture was added to confluentmonolayers of either endothelial cells (e.g. HUVEC cells) or fibroblastsin 96 well flat bottom plates and incubated at 37° C. for two days. Thesupernatant was discarded, the cells were fixed with cold methanol andstained with a mixture of mouse monoclonal antibodies to hCMV earlyantigens, followed by a fluorescein-labelled goat anti mouse Ig. Theplates were analyzed using a fluorescence microscope. In the absence ofneutralising antibodies the infected cells were ˜1000/field, while inthe presence of saturating concentrations of neutralising antibodies theinfection was completely inhibited. The neutralising titer is indicatedas the concentration of antibody (μg/ml) that gives a 50% reduction ofhCMV infection.

Table 2 shows that three different types of antibodies have beenidentified. Those that can neutralise infection of fibroblasts, thosethat can neutralise infection of endothelial cells and those that canneutralise infection of both. This agrees with previous data thatdifferent proteins are responsible for tropism towards a particular celltype [7]. In addition to neutralisation of endothelial cells, 1F11 and2F4 were observed to neutralise infection of epithelial cells such asretinal cells, and dendritic cells (data not shown).

TABLE 2 50% neutralisation (μg/ml) Clone Specificity FibroblastsEndothelial cells 1F11 UL130/UL131A * 0.001 2F4 UL130/UL131A * 0.003 5A2UL130/UL131A * 0.002 9A11 UL130/UL131A * 0.001 7H3 gB 2 * 10C6 gB 0.30.3 5F1 gB 0.3 0.3 6B4 gB 0.5 * Cytotec{circumflex over ( )} 5000 50Donor's Serum 33 1 * no neutralisation at the highest concentrationtested (i.e. >2 μg/ml). {circumflex over ( )}Cytotect (Biotest) is apool of hCMV hyperimmune IgG.

Some antibodies neutralized infection of both fibroblasts andendothelial cells at IgG concentrations ranging from 0.3 to 0.5 μg/ml.Other antibodies (1F11, 5A2, 9A11 and 2F4) failed to neutralize hCMVinfection of fibroblasts, but neutralized the infection of endothelialcells and did so at extremely low concentrations ranging from 0.001 to0.004 μg/ml (more than 1000 fold more potent than previously knownantibodies).

Note that since the initial characterization, it has been determinedthat 5F1 binds to an epitope of gB rather than gH. This is consistentwith the results which demonstrate that blocking gB allowsneutralization of infection of fibroblasts as observed for 7H3, 1006 and6B4.

EXAMPLE 2 Identification of the Target Antigens Recognised by theMonoclonal Antibodies

Human MRC-9 fibroblasts were infected with a clinical hCMV isolate.After 3 days the cells were metabolically labeled with ³⁵S Methionineand Cysteine. After preclearance of the lysate human monoclonalantibodies 1F11 and 2F4 were added and immunocomplexes were precipitatedby the addition of Protein A beads and resolved on SDS-PAGE (FIG. 1). Ahuman monoclonal IgG antibody with irrelevant specificity was used asnegative control. The results show that human monoclonal antibodies 1F11and 2F4 precipitate complexes of CMV proteins.

To map the specificity of the human monoclonal antibodies expressionvectors encoding hemagglutinin (HA)-tagged UL128Δ1-27, UL130Δ1-25 andUL131AΔ1-18 hCMV proteins lacking signal peptides were constructed.HEK293T cells were transfected with these vectors alone or incombination. After 36 h, cells were fixed, permeabilized and stainedwith an anti-IIA antibody (to control for efficiency of transfection)and with monoclonal antibodies followed by a goat anti-human IgG. AHuMab IgG with irrelevant specificity was used as negative control. FIG.2A shows that the human monoclonal antibodies 1F11 and 2F4 recognise aconformational epitope made by UL130 and UL131A gene products. FIG. 2Bshows that the human monoclonal antibodies 5A2 and 9A11 recognise aconformational epitope made by the UL130 and UL131A gene products.

CONCLUSIONS

The above results define two human monoclonal antibodies that arecapable of neutralizing with high potency and selectivity the hCMVinfection of human endothelial cells. To identify the epitoperecognized, the antibodies were tested for their capacity toimmunoprecipitate proteins from hCMV infected cells (FIG. 1). Human Mabs1F11 and 2F4 precipitated several proteins with apparent molecularweights of ˜15, 33-35 and ˜100 KDa. These patterns are compatible withthe precipitation of a complex containing gH, gL and UL128, UL130 andpossibly UL131A.

To better define the target of these antibodies we characterized theircapacity to stain HEK293T cells transfected with vectors encodingHA-tagged UL128, UL130 and UL131A. As shown in FIG. 2A, 1F11 and 2F4stained only cells coexpressing UL130 and UL131A, suggesting that theyrecognize a conformational epitope determined by the two gene products.This conclusion is supported by the fact that these antibodies do notreact in a western blot with lysates of infected or transfected cellsrun under reducing, denaturing, conditions (data not shown).

Similar results were observed for 5A2 and 9A11. FIG. 2 b shows thatthese antibodies stained only cells coexpressing UL130 and UL131A,suggesting that they recognize a conformational epitope determined bythe two gene products.

EXAMPLE 3 Further Identification of the Target Antigens Recognised bythe Monoclonal Antibodies

To map specificities of human monoclonal antibodies neutralizinginfection of fibroblasts an expression vector encoding full length gBwas constructed. HEK293T cells were transfected with this vector. After36 h, cells were fixed, permeabilized and stained with human monoclonalantibodies (HuMab) followed by goat anti-human IgG. FIG. 4 shows thatmonoclonal antibodies 7H3, 10C6, 5F1, and 6B4 (but not an IgG antibodyof an irrelevant specificity) specifically stained cells transfectedwith gB, indicating that they recognize an epitope of gB. Of note, themonoclonal antibodies 10C6, 5F1 and 6B4 neutralise infection offibroblasts and endothelial cells, whereas the monoclonal antibody 7H3neutralises the infection of fibroblasts (but not of endothelial cells).This notion suggests that the monoclonal antibodies 10C6, 5F1, and 6B4bind to a functional epitope of gB that is distinct from the epitopebound by the monoclonal antibody 7H3.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

REFERENCES The Contents of which are Hereby Incorporated by Reference

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The invention claimed is:
 1. A composition comprising an isolatedantibody, or an antigen binding fragment thereof, comprising the CDR1,CDR2 and CDR3 sequences of the heavy and light chain region sequencesset forth respectively in SEQ ID NO:s 17 and 18, wherein the compositionis in lyophilized form.
 2. A composition comprising an isolatedantibody, or an antigen binding fragment thereof, comprising heavy chainvariable region CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:11, 12 and 13, respectively, and light chain variable region CDR1, CDR2,and CDR3 sequences set forth in SEQ ID NOs: 14, 15 and 16, respectively,wherein the composition is in lyophilized form.
 3. The composition ofclaim 1, comprising the heavy and light chain variable region sequencesset forth in SEQ ID NOs: 17 and 18, respectively.
 4. The composition ofclaim 1, that is specific for a complex of human cytomegalovirus (hCMV)proteins UL130 and UL131A.
 5. The composition of claim 1, which inhibitsinfection of epithelial cells, wherein the concentration of antibodyrequired for 50% inhibition of hCMV is 10 μg/ml or less.
 6. Thecomposition of claim 5, wherein the concentration of antibody requiredfor 50% inhibition of hCMV is 0.2 μg/ml or less.
 7. The composition ofclaim 5, wherein the concentration of antibody required for 50%inhibition of hCMV is 0.1 μg/ml or less.
 8. The composition of claim 5,wherein the concentration of antibody required for 50% inhibition ofhCMV is 0.01 μg/ml.
 9. The composition of claim 1, wherein the antibodyis a human antibody, a monoclonal antibody, a single chain antibody,Fab, Fab′, F(ab′)2, Fv or scFv.
 10. The composition of claim 1, furthercomprising a second antibody, or an antigen binding fragment thereof,which inhibits hCMV infection.
 11. The composition of claim 10, whereinthe second antibody binds to an hCMV gB protein or gH protein.
 12. Amethod of inhibiting hCMV infection in a subject, comprisingadministering via injection an effective amount of the composition ofclaim 1, wherein the composition is reconstituted prior to injectionand, wherein hCMV infection is inhibited.
 13. A method of inhibitinghCMV infection in an isolated cell, comprising contacting the cell withan effective amount of the composition of claim 1 followingreconstitution of the composition, wherein hCMV infection is inhibited.14. The method of claim 12, further comprising the step of administeringa second composition comprising a second antibody, or an antigen bidingfragment thereof, which inhibits hCMV infection, wherein thecompositions are administered simultaneously or sequentially.
 15. Themethod of claim 13, further comprising the step of contacting the cellwith a second composition comprising a second antibody, or an antigenbiding fragment thereof, which inhibits hCMV infection, wherein the cellis contacted by the compositions simultaneously or sequentially.